Methods and compositions are described of determining if proteins have been transferred to an array, and for characterization of the transferred (deposited) proteins.
The protein chip is a tool which is used in drug discovery and proteomics (i.e., the study of protein expression profiles and protein-protein interactions), not only for understanding basic cellular processes at the protein level, but also as a platform for next generation clinical and medical diagnostic devices or systems. Continued advances in protein chip technology, however, have been hindered by the very nature of proteins themselves. Variations in molecular weight, hydrophobicity, charge, stability, post-translational modifications (PTM), and accessibility of reactive side chains for immobilization are a few examples of protein heterogeneity that directly affect microarray fabrication, reproducibility, and multiplexed analyses. Variations in liquid handling, protein aggregation, buffer ionic strength, and sample viscosity likewise lead to significant variations in protein array manufacture and quality, necessitating the development of thorough quality assurance and quality control (QA/QC) methods for routine manufacturing practice.
As with nucleic acid arrays, dye incorporation can be used to confirm volume transfer from a printing system to a microarray substrate, but dye incorporation does not quantify the amount of protein transferred and functionally immobilized on the array. Likewise, a single, labeled, and co-deposited protein (analogous to an internally-doped oligonucleotide probe) may be a poor proxy for deposition/immobilization efficiency for proteins of substantively different physicochemical properties. While direct labeling of capture probes prior to deposition is possible for nucleic acids because hybridization is largely unaffected by 3′ or 5′ terminal fluors, equivalent protein labeling procedures may not only affect the physicochemical properties of the protein, but also consume reactive side chains necessary for the covalent immobilization process.
Creating functional protein arrays typically requires a sequenced genome, a well-characterized protein expression system, and labor-intensive expression and purification methods. In addition, the majority of proteins expressed in vitro lack PTMs that may be required for proper function or interaction, and expression systems for outer membrane proteins are still in their infancy. Practical difficulties associated with protein expression and purification are, therefore, obvious impediments to protein array manufacture and use, resulting in a number of new methods for generating protein array content.
A recently described two-dimensional liquid phase separation technique (PF2D) creates protein expression profiling maps. The PF2D fractions may create comprehensive, proteome-scale, functional protein arrays. Because proteins are generated in vivo by the organism of interest, a sequenced genome is not required in order to generate protein content reproducibly, and the resulting fractionated proteins retain all PTMs intact. Due to the uncharacterized nature of PF2D protein fractions, however, methods for confirming the successful transfer and abundance of uncharacterized PF2D fractionated proteins within microarray features becomes critical for subsequent assay development and interaction assay data interpretation.